Process for preparing purified dimer of bone-derived factor

ABSTRACT

A process for preparing a purified dimer of a bone-derived factor, which comprises subjecting an inclusion body of a bone-derived factor produced by genetic engineering to the following steps a) to e) in sequence: 
     a) the step of treating an inclusion body of a bone-derived factor with a unfolding agent to prepare a solubilized monomer; 
     b) the step of treating the solubilized monomer with a refolding solution to prepare a dimer; 
     c) the step of subjecting the refolded dimer to ultrafiltration and solvent replacement; 
     d) the step of subjecting the dimer solution prepared above to isoelectric precipitation; and 
     e) the step of subjecting the isoelectrically precipitated dimer to reverse-phase chromatography.

This application is a 371 of PCT/JP97/04784 filed Dec. 24, 1997.

FIELD OF THE INVENTION

This invention relates to a process for the production of a purifieddimeric bone morphogenetic factor. More particularly, it is concernedwith a process for the production of a dimeric bone morphogeneticfactor, characterizing in acquiring a purified dimeric bonemorphogenetic factor from an inclusion body of a bone morphogeneticfactor produced by means of a genetic engineering technology.

BACKGROUND OF THE INVENTION

A proteinaceous bone morphogenetic factor was discovered to be presentin the bone matrix (Science 150, pp.893-899, 1965) and was named as“bone morphogenetic protein” (hereinafter abbreviated as BMP). Recently,cloning of plural BMP-related genes has been attempted and it has beenfound that all of them belong to the transforming growth factor-β(hereinafter abbreviated as TGF-β) superfamily. Recombinants of some ofthese factors have been produced by means of a genetic engineeringtechnology and they have been confirmed to have a bone morphogeneticactivity, from which their application to the treatment of bone diseasesis expected.

Of these factors, the human GDF-5 (MP52) recently discovered andbelonging to the human BMP family (Biochem. Biophys. Res. Commun., 204,pp. 646-652, 1994) has been confirmed by animal tests to be effective asa bone morphogenetic factor, while it has been technically reviewed tocarry forward the large-scale production thereof by expression usingrecombinant Escherichia coli (E. coli). However, when expressed in alarge scale in E. coli and others, for instance, when the protein isproduced at an amount of several grams per liter of cultured broth, thedesired protein generally tends to form an inactive and insolubleinclusion body. This inclusion body is monomeric and, in order to obtaina dimer which is active as a bone morphogenetic factor, the inclusionbody must be solubilized, renatured to a dimer of an original structure(the procedure generally called “refolding”), separated and purified toobtain the desired protein.

The active form of MP52 has the following or the like problems;

1) because of its low solubility in an aqueous solution, it should behandled in the presence of a denaturing agent or under acidicconditions,

2) the protein used for separation tends to nonspecifically adsorb ontoa resin for liquid chromatography, and

3) the surfactant essential for refolding tends to disturb separation,

and thus it has been very difficult to establish a process for thepurification thereof.

The purification process recently developed for solving theabove-mentioned problems (WO 96/33215) successful in obtaining a singleactive form of MP52 comprises the following steps;

1. solubilizing an inclusion body by a denaturing agent,

2. separation by ion exchange chromatography,

3. sulfonation,

4. separation by gel filtration chromatography,

5. refolding,

6. recovery by isoelectric precipitation, and

7. separation by reverse-phase chromatography.

However, the above process if scaled up industrially has encountered thefollowing and the like problems;

1) a large amount of a denaturing agent is used in order to solubilizethe MP52 inclusion body, whereby modification of the protein (forexample, carbamylation reaction in the case of urea) may be induced,

2) an expensive resin for chromatography, especially, for gel filtrationchromatography, such as Sephacryl S-200HR or Superdex 200 pg (allavailable from Pharmacia Biotech) is used in a large amount,

3) a reagent used in refolding, inter alia, CHAPS and oxidizedglutathione essential for dimerization reaction is extremely expensive,and

4) when isoelectric precipitation is carried out, the dilution isnecessary to decrease the concentration of detergent, thus the volume ofthe solution is increased.

DISCLOSURE OF THE INVENTION

An object of this invention is to solve the above-mentioned problems,i.e.,

1) to use a denaturing agent in an amount as low as possible;

2) to use a chromatography resin in an amount as low as possible;

3) to replace the reagent used for refolding by other inexpensive onesand to simplify concomitant procedures with refolding,

4) to decrease the volume of the solution by removing a detergentselectively; that is, to considerably shorten the process time.

The present inventors have made feasible a simplification of thepurification steps by solubilizing an inclusion body extracted from E.coli in the presence of a denaturing agent, conducting a directrefolding according to a dilution procedure and then subjecting anultrafiltration substititing the refolding solution. This procedureappears to be similar to the first step of a process for the productionof human insulin from E. coli (EP 600372A1). However, since a bonemorphogenetic factor is different in properties from a soluble proteinsuch as human insulin, it was difficult to apply the process for theproduction of insulin as depicted above in case of a bone morphogeneticfactor. MP52 (active form) dimerized as depicted above has a lowsolubility and tends to adsorb onto a chromatographic resin, thus in thelarge-scale production, the ion exchange chromatography or hydrophobicchromatography used for human insulin or the gel filtrationchromatography used in the above-mentioned WO 96/33215 could not beapplied. When an ion exchanger (SP Sepharose FF, Pharmacia Biotech) isused, for example, MP52 is not completely eluted because of its strongadsorption onto the resin, even if a denaturing agent and a maximum saltconcentration is used. When gel filtration (Sephacryl S-200HR, PharmaciaBiotech) is used, a strong adsorption of the protein onto a resin occurseven if a denaturing agent is used, causing an excessively broadenedfractionation range and thus a very poor separation. Further, propertiesof the resin is altered by influence with of a surfactant such as CHAPS,which leads to loss of reproducibility. This is also applicable to theelution with an acidic solution in which MP52 is soluble. In conclusion,it is not feasible to make use of the original properties of the resin.

As explained above, it has become apparent that the purification of thedesired protein in large-scale production can not be accomplishedaccording to a general chromatographic means using aqueous system.Reverse-phase chromatography using organic solvent is the only meansthat could be utilized. In view of this, it was necessary to develop apurification means wherein many columns are not used. As purificationmeans other than using columns, a fractionating method by ammoniumsulfate seemed promising. However, since it had low purificationefficiency and led to unnecessarily low yield, its use was cast aside.In addition, isoelectric precipitation procedure by pH adjustment wasadopted, but prior to the actual procedure, an ultrafiltration procedureto remove a surfactant, CHAPS, was carried out which enabled theperformance of isoelectric precipitation without increasing the volumeof the solution. Conventionally, when the solution contained CHAPS, theprotein solubility was high and no precipitation occurred. Therefore, adilution was necessary to decrease the concentration of CHAPS, but aresultant extensive increase in solution volume has been a problem inprocess development.

This invention is directed to a process for the production of a purifieddimeric bone morphogenetic factor, characterizing in subjecting aninclusion body of a bone morphogenetic factor produced by means of agenetic engineering technology to the following steps a)-e) in order,thereby producing a dimeric bone morphogenetic factor;

a) treating an inclusion body of a bone morphogenetic factor with adenaturing agent to obtain a solubilized monomer,

b) treating the solubilized monomer with a refolding solution to obtaina dimeric bone morphogenetic factor,

c) treating the dimeric bone morphogenetic factor by ultrafiltration andsubstitution of solvent,

d) subjecting the dimeric bone morphogenetic factor in solvent thussubstituted to isoelectric precipitation, and

e) subjecting the dimeric bone morphogenetic factor thus precipitated toreverse-phase chromatography.

The inclusion body of a bone morphogenetic factor produced by means of agenetic engineering technology is preferably the one expressed in E.coli by means of a genetic engineering technology.

When a bone morphogenetic factor is expressed in E. coli, the cells aresuspended in a buffer, homogenized by means of a homogenizer andcentrifuged to recover an inclusion body. The inclusion body is washedwith a buffer containing a detergent, for example, Triton X-100, orurea, for more than 3 times and centrifuged to obtain an inclusion bodyof primary purification.

The step in which an inclusion body of a bone morphogenetic factor istreated with a denaturing agent to give a solubilized monomer may becarried out by adding the inclusion body to a solution containing thedenaturing agent and dissolving by stirring. For the solution containinga denaturing agent, any of those publicly known such as 8 M urea or 6 Mguanidine-HCl and others in 50 mM glycine-NaOH buffer (pH 10.7) may beused.

The step in which a solubilized monomer is treated with a refoldingsolution to give a dimer, is carried out by diluting the proteinsolution obtained above with a refolding solution so as to provide afinal protein concentration of 0.1 to 5.0 mg/mL, preferably 2.4 mg/mL.Although dilution has been hitherto made so as to provide a finalconcentration of a denaturing agent to 1M or less, it is preferable inthis invention that the dilution be made so as to provide a finalconcentration of a denaturing agent to 1-4 M, particularly, 2.4 M, sothat aggregation and precipitation of proteins may be prevented with animproved yield. For the refolding solution, any of those publicly knownin the prior art such as surfactants, e.g., cholic acid or itsderivatives such as3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), taurocholic acid or a salt thereof, taurodeoxycholic acid or asalt thereof, and preferably,3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS); EDTA;a combination of a reducing agent such as mercaptoethanol ordithiothreitol (DTT) with oxidized glutathione or a buffer containingcysteine or the like may be used. The advantage of using cysteine aloneis that it is not necessary to use generally expensive oxidizing reagentand that it can decrease its amount of use. Therefore, it is expected tosimplify the process steps and to save the cost for reagents.

The reagent contained in the refolding solution other than abovedescribed is that having a guanidino group which also prevents a proteinaggregation and precipitation and increases the yield. Morespecifically, guanidine hydrochloride or arginine hydrochloride(Arg.HCl), preferably 0.5 M of Arg.HCl is added to the refoldingsolution in advance. The effect of the addition of Arg.HCl is shown inTable 1.

TABLE 1 Protein concentration (g/L) MP52 dimer (g/L) 0.8* + 0.5 MArg.HCl 0.37 1.6 + 0.5 M Arg.HCl 0.62 2.4 + 0.5 M Arg.HCl 0.98 3.2 + 0.5M Arg.HCl 0.95 2.4 without Arg.HCl protein aggregation occurred *Proteinconcentration of 0.8 g/L was the upmost limit for the proteinconcentration without Arg.HCl.

As shown in Table 1, without Arg.HCl, protein aggregation andprecipitation occurred in the refolding solution. However, by addingArg.HCl, the amount of protein that can be treated per refoldingsolution without aggregation can be increased by 2.7 fold (i.e. 0.98 g/Las opposed to 0.37 g/L). As for a buffer, those buffers using phosphateor Tris-HCl may be used, but an Arg-NaOH buffer of pH 8-10, particularlypH 8.9, is preferred.

The ultrafiltration step in which the refolded dimer is concentrated, iscarried out by using a molecular weight cut-off membrane filter of10,000 such as PSU 10K (Sartorius) and the CHAPS concentration islowered by substituting with an acid solution, such as 0.2% phosphoricacid solution.

The step in which the solution of the dimer substituted isisoelectrically precipitated, is carried out by adding alkali solutionsuch as NaOH to the dimer solution adjusting pH value to 7.4 toselectively precipitate a bone morphogenetic factor. After the pHadjustment, the solution is allowed to stand for one hour or more,centrifuged or filtered to remove the supernatant and the precipitate isdissolved in an acid solution such as 50 mM citric acid, 0.2% phosphoricacid or 0.05% trifluoroacetic acid solution.

The step in which the isoelectrically precipitated dimer is subjected toreverse-phase chromatography, is carried out by subjecting the acidicsolution obtained above to high-performance liquid chromatography andeluting with 0-50% gradients of organic solvent such as isopropanol,acetonitrile or ethanol to recover the fractions of a dimeric bonemorphogenetic factor. As resin for high-performance liquidchromatography, a polymeric resin such as SOURCE 15 RPC (6 cmφ×20 cm,manufactured by Pharmacia Biotech) is used.

The bone morphogenetic factor to be used in this invention is preferablya bone morphogenetic factor having a single molecular weight selectedfrom the group consisting of MP52, BMP-2, BMP-4, BMP-6 and BMP-7. As anexample, the E. coli strain having introduced therein cDNA encoding ahuman MP52 precursor (specifically, the E. coli having introducedtherein a plasmid ligated with a codon encoding methionine at the5-primer terminus in MP52-sequence of 119 residues of which theN-terminal alanine of mature human MP52 is deleted) is incubated toproduce mature monomeric MP52 as inclusion body in large amounts andusing the present process, mature MP52 is obtained with high purity fromthis inclusion body.

EXAMPLE

This invention will be more specifically explained hereinbelow by way ofexamples, which are not construed to limit the invention. The proceduresfrom (2) to (4) were carried out in a low temperature chamber at 4° C.,considering stability of the protein. Each step will be fully explainedbelow.

(1) Fermentation of Human MP52 and Primary Purification of InclusionBody

The MP52-producing E. coli obtained in the same manner as described inExample 2 of WO 96/33215 was precultivated in a modified SOC medium andthen the precultivated broth was inoculated into 100 L of productionmedium. For induction, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG)was added at an early logarithmic growth phase and fermentation wascontinued at 32° C. until OD₅₅₀=150. After that, cells were harvested,cells were suspended in a buffer containing 25 mM Tris-HCl (pH 7.3) and10 mM EDTA-4Na, homogenized by means of a homogenizer (manufactured byManton Gaulin) and centrifuged to recover an inclusion body. Theinclusion body was washed with a buffer containing 1 M urea as adetergent and centrifuged to obtain an inclusion body with primarypurification applied.

(2) Solubilization of Inclusion Body and Refolding

One hundred g (wet weight) of the inclusion body obtained above wassolubilized by stirring in 300 mL of 50 mM glycine-NaOH buffercontaining 8 M urea and 5 mM ethylenediamine-tetraacetate (pH 8.9)(protein concentration being about 18 mg/mL). Refolding was performed bydiluting the inclusion body solution with a refolding buffer [0.5 MArg-NaOH (pH 8.9), 4.8 mM cysteine hydrochloride monohydrate, 0.5 Msodium chloride, 20 mM CHAPS] to 6.7 times volume (proteinconcentrationbeingabout 2.4 mg/mL). The mixture as such was allowed tostand at 4° C. for about 20 hours.

(3) Purification of MP52 after Refolding (Ultrafiltration)

MP52 after completion of the refolding reaction was concentrated 5 foldby using a membrane filter of 10,000 cut-off molecular weight (PSU 10K,Sartorius) and the solution was diluted and substituted by 5 fold volumewith 0.2% phosphoric acid solution. By repeating the procedure threetimes, the CHAPS concentration is diluted theoretically by 100 fold ormore.

(4) Purification of MP52 after Refolding (Isoelectric Precipitation)

Isoelectric precipitation was performed by adding NaOH solution to thesubstituted refolding solution adjusting the pH value to 7.4. Thesolution became cloudy, and then it was allowed to stand for one hour ormore. Then it was centrifuged (10,000 g×15 min) and the precipitate wasrecovered. The precipitate was dissolved in 0.2% phosphoric acidsolution.

(5) Purification of MP52 after Refolding (Reverse-Phase Chromatography)

MP52 dissolved in the phosphoric acid solution was separated by means ofreverse-phase chromatography. A high-performance liquid chromatographicsystem using SOURCE 15 RPC (6 cmφ×20 cm, Pharmacia Biotech) as resin wasoperated and eluted with 0-50% ethanol gradient to recover the fractionscontaining dimeric MP52.

According to the above-mentioned purification process, we have succeededin recovering an active form of MP52 in high yield as shown in Table 2.An amount of MP52 in the purification step was determined byquantification of scanned CBB-stained-electrophoresis gel image.

TABLE 2 Step Amount of MP52 (g) Yield (%) Solubilization 5.4 100 Refolding 2.2 41 Ultrafiltration 1.6 30 Isoelectric precipitation 1.5 29Reverse-phase chromatography 1.1 21

One of the advantages of the purification process in this invention isan effective reduction of the purification cost. According to apreliminary calculation, the total process cost may be reduced to about½ per protein as compared with those in WO 96/33215. Therefore, thepresent purification process can be very useful in industrialization.

According to the present process, a dimeric bone morphogenetic factorhaving a single molecular weight can be efficiently produced in a largeamount and more inexpensively, as compared with the prior art process.

We claim:
 1. A process for the production of a purified dimeric bonemorphogenetic factor which comprises subjecting an inclusion body of abone morphogenetic factor to the following steps a)-c) in order, therebyproducing the dimeric bone morphogenetic factor; a) introducing apolynucleotide encoding a bone morphogenetic factor into E. coliexpressing said bone morphogenetic factor in the form of an inclusionbody in E. coli, recovering (and washing) said inclusion body andtreating it with a denaturing agent to obtain a solubilized monomer, b)treating the solubilized monomer without purification directly with arefolding solution in a final protein concentration from 1.6 mg/ml up to5 mg/ml to obtain a dimeric bone morphogenetic factor, c) subjecting thedimeric bone morphogenetic factor to purification.
 2. The process ofclaim 1, wherein the refolding solution has a final concentration ofsaid denaturing agent of 1 M-4 M.
 3. The process for the productionaccording to claim 1, wherein said refolding solution is a buffercomprising cysteine or salt thereof, sodium chloride at a concentrationof 0.1 to 1.5 M, and cholic acid or its derivatives at a concentrationof 5 to 100 mM and a pH in the range of 8-10.
 4. The process for theproduction according to claim 3, wherein said refolding solution isfurther comprising a guanidine or arginine or a salt thereof.
 5. Theprocess for the production according to claim 1, wherein said bonemorphogenetic factor is a bone morphogenetic factor selected from thegroup consisting of MP52, BMP-2, BMP-4, BMP-6 and BMP-7.
 6. The processof claim 1 wherein the dimeric bone morphogenetic factor is purified byultrafiltration, isoelectric precipitation and reverse-phasechromatography.